Alphaviruses such as Chikungunya virus (CHIKV) and Venezuelan equine encephalitis virus (VEEV) are important emerging human pathogens known to cause explosive epidemics. These viruses are responsible for outbreaks of viral encephalitis or painful recurring arthralgia in humans. Each year they have the potential to spread from a mosquito or avian reservoir to new regions. Large sums have been invested in vector surveillance and symptom management, but therapeutics and vaccines that protect against alphavirus infection have had limited success. Our group studies the pathogenesis of alphaviruses in vivo, including CHIKV and Sindbis virus (SINV). These kinds of studies can contribute to the design of more effective therapeutics and vaccines through an enhanced understanding of the molecular events that occur during infection. Our LONG-TERM GOAL is to understand the exact mechanisms of alphavirus interactions with their hosts. In this proposal, we investigate the interaction between SINV and two related receptors, mammalian NRAMP 1 and 2. Infection of mammalian cells by the alphavirus Sindbis virus (SINV) requires natural resistance-association macrophage protein 2 (NRAMP2) for entry. However, this requirement has never been evaluated in vivo. Further, the interaction between SINV and the closely related and functionally similar NRAMP1 protein has never been evaluated. Therefore, our OBJECTIVE in this study is to define the consequence of NRAMP2 utilization on SINV pathogenesis and to determine whether a similar role exists for NRAMP1. We HYPOTHESIZE that the mammalian NRAMP proteins are important determinants of SINV tropism and pathogenesis. To achieve our objective, we have proposed the following aims. In SPECIFIC AIM 1, we will analyze the role of NRAMP2 in host susceptibility to disease and virus tropism. We will achieve this aim using tissue- specific and inducible NRAMP2 knockout mouse models. In SPECIFIC AIM 2, we will investigate whether NRAMP1 functions as an additional entry receptor during SINV infection. We will use an in vitro model to overexpress NRAMP1 and define the relative utilization of mouse or human NRAMP 1 versus NRAMP2 by SINV. These studies will define the precise impact of mammalian NRAMP expression on SINV infection and disease. Successful completion of these aims could indicate that the NRAMP proteins are appropriate and novel therapeutic targets. The proposed study provides the applicant with an excellent source of training in virus-host interactions that impact alphavirus pathogenesis.